The present invention relates to a separating agent for enantiomeric isomers, in particular, a separating agent used for separating enantiomeric isomers in liquid chromatography. More particularly, the present invention relates to a separating agent for enantiomeric isomers that can enantiomerically resolve a broad range of chiral compounds with high separation factors in the analysis of pharmaceuticals, foods, agricultural chemicals, fragrants and the like and a method of evaluating the ability of recognizing asymmetry of such a separating agent.
Many organic compounds have isomers that have the same physical and chemical properties, such as boiling point, melting point and solubility but show a difference in a physiological activity, i.e., enantiomeric isomers. This difference in physiological activity between the isomers is attributable to the following. In most cases, proteins and carbohydrates that compose a living body of a living organism are composed only of the one of enantiomeric isomers so that they show a difference in the manner of action to the other kinds of enantiomeric isomers, resulting in a difference in the physiological activity. This can be compared to a difference in easiness (difference in physiological activity) of wearing of a glove for left hand (i.e., a living organism as an enantiomerically active substance) between the right hand and the left hand (respective enantiomeric isomers that act).
In particular, in the field of pharmaceutical preparations, in many cases, there are significant differences in medical property and toxicity between the two enantiomeric isomers. Therefore, in the Guideline for the Production of Pharmaceuticals, the Ministry of Health, Labor and Welfare describes a policy for making a sharp distinction between enantiomeric isomers saying xe2x80x9cwhen the drug of interest is a racemic modification, it is desirable to preliminarily study absorption, distribution, metabolism and excretion kinetics of each enantiomeric isomer.xe2x80x9d
Since enantiomeric isomers have completely the same physical and chemical properties, such as boiling point, melting point, and solubility as previously stated, they cannot be analyzed by ordinary separation means. For this reason, extensive studies have been made on techniques for separating enantiomeric isomers that analyze a wide variety of enantiomeric isomers conveniently and with high precision. As a result, as an analytical technique that meets these requirements, an enantiomeric resolution method by high performance liquid chromatography (HPLC), in particular an enantiomeric resolution method by using a chiral column for HPLC has been developed. The chiral column referred to herein uses an asymmetry recognition agent itself or a chiral stationary phase composed of an asymmetry recognition agent supported on a suitable carrier.
For example, enantiomerically active poly (triphenylmethyl methacrylate) (cf., JP 57-150432 A), cellulose or amylose derivatives (Y. Okamoto, M. Kawashima and K. Hatada, J. Am. Chem. Soc., 106, 5357, 1984), ovomucoid, which is a protein (JP 63-307829 A) and the like have been developed.
It has been known that among many chiral stationary phases for HPLC, an enantiomeric resolution column having supported cellulose or amylose derivative on silica gel has high asymmetry recognition ability to an extremely wide variety of compounds. Furthermore, in recent years, studies on a liquid chromatographic fractionation method for fractionating enantiomerically active substances on an industrial scale including a chiral stationary phase for HPLC and a simulated moving bed method, which is a continuous liquid chromatographic fractionation method in combination have been developed (Pharm Tech. Japan, 12, 43 (1996)).
For example, enantiomerically active poly (triphenylmethyl methacrylate) (cf., JP 57-150432 A), cellulose or amylose derivatives (Y. Okamoto, M. Kawashima and K. Hatada, J. Am. Chem. Soc., 106, 5357, 1984), ovomucoid, which is a protein (JP 63-307829 A) and the like have been developed.
It has been known that among many chiral stationary phases for HPLC, an enantiomeric resolution column having supported cellulose or amylose derivative on silica gel has high asymmetry recognition ability to an extremely wide variety of compounds. Furthermore, in recent years, studies on a liquid chromatographic fractionation method for fractionating enantiomerically active substances on an industrial scale including a chiral stationary phase for HPLC and a simulated moving bed method, which is a continuous liquid chromatographic fractionation method in combination have been developed (Pharm Tech. Japan, 12, 43 (1996)).
In the case of chiral stationary phase for HPLC used as analysis means, complete separation of two enantiomeric isomer peaks in a short analysis time gives a full satisfaction. However, in order to further increase fractionation productivity, a liquid chromatographic fractionation method as production means has been required to not only completely separate a compound as a target of fractionation but also further separate two enantiomeric isomer peaks of the target compound; that is, a chiral stationary phase having a value of separation factor xcex1 as high as possible has been desired.
Under the circumstances, various contrivances have been made to more fully develop the asymmetry recognition ability of the chiral stationary phase including an enantiomerically active polymer compound such as, for example, a polysaccharide derivative as an asymmetry recognition agent to obtain a further increased value of separation factor xcex1. Under the present conditions, however, there are no evaluation methods for high asymmetry recognition ability other than those that use an HPLC measurement in reality. Accordingly, a simpler and easier method of evaluating asymmetry recognition ability has been demanded.
JP-A 2-289601 discloses a separating agent comprising a polysaccharide derivative having xe2x80x94COxe2x80x94NHR for OH.
The present invention has been achieved under the above-mentioned circumstances. That is, an object of the present invention is to provide a simpler and easier method of evaluating asymmetry recognition ability. Another object of the present invention is to provide a separating agent for enantiomeric isomers having higher asymmetry recognition ability by using the evaluation method.
As a result of extensive studies for achieving the above-mentioned objects, the inventors of the present invention have now found that those separating agents that cause polymer compounds which have been supported therein to exhibit exothermic peaks before the polymer compounds reach their decomposition temperatures in a differential thermal calorimetric curve obtained in a heat elevation process in differential scanning calorimetry (DSC) have high values of separation factors xcex1 of enantiomeric isomers, thereby achieving the present invention.
Therefore, the present invention provides a separating agent for enantiomeric isomers, comprising an enantiomerically active polymer compound supported thereon, wherein the polymer compound has an exothermic peak before a decomposition temperature of the polymer compound supported is reached in a differential calorimetric curve obtained in a process of temperature elevation in differential scanning calorimetry (DSC) on the separating agent.
It then provides a method of evaluating asymmetry recognition ability of a separating agent for enantiomeric isomers, comprising: performing a differential scanning calorimetry (DSC) of the separating agent for enantiomeric isomers having supported thereon an enantiomerically active polymer compound to obtain a differential calorimetric curve in a process of temperature elevation therein; and observing presence or absence of an exothermic peak of the polymer compound in the differential calorimetric curve before a decomposition temperature of the supported polymer compound is reached.
Hereinafter, the present invention will be described in detail by embodiments. However, the present invention is not limited thereto.
The enantiomerically active polymer compounds used in the present invention include polysaccharide derivatives, enantiomerically active polyamides, enantiomerically active polyesters, enantiomerically active polyamino acids, enantiomerically active polyethers, polymers having bound thereto enantiomerically active compounds, proteins, and modified proteins and complexes of these. In particular, polysaccharide derivatives or complexes thereof are suitably used.
The polysaccharide derivatives used in the present invention can be obtained by reacting a polysaccharide with a compound having a functional group that is reactive with the hydroxyl groups of the polysaccharide.
The polysaccharide may be any polysaccharide, being a synthetic or natural one or a modified natural one. The polysaccharide has preferably a high regularity in the manner of binding between saccharides. Examples of the polysaccharide include xcex2-1,4-glucan (cellulose), xcex1-1,4-glucan (amylose or amylopectin), xcex1-1,6-glucan (dextran), xcex2-1,6-glucan (pustulan), xcex2-1,3-glucan (for example, curdlan, schizophyllan, etc.), xcex1-1,3-glucan, xcex2-1,2-glucan (crown gall polysaccharide), xcex2-1,4-galactan, xcex2-1,4-mannan, xcex1-1,6-mannan, xcex2-1,2-fructan (inulin), xcex2-2,6-fructan (levan), xcex2-1,4-xylan, xcex2-1,3-xylan, xcex2-1,4-chitosan, xcex1-1,4-N-acetylchitosan (chitin), pullulan, agarose and alginic acid. Also, starches containing amylose are included therein. Among these polysaccharides, it is preferable to use cellulose, amylose, xcex2-1,4-xylan, xcex2-1,4-chitosan, chitin, xcex2-1,4-mannan, inulin, curdlan, etc. which can be easily obtained as highly pure polysaccharides, still preferably cellulose and amylose.
It is preferable that such a polysaccharide has a number-average degree of polymerization (i.e., the average number of pyranose or furanose rings per molecule) of at least 5, still preferably at least 10. From the viewpoint of handling properties, it is preferable that the number-average degree of polymerization thereof is not more than 1,000, though the upper limit thereof is not particularly defined.
The compounds having functional groups capable of reacting with the hydroxyl groups of the polysaccharide may be isocyanic acid derivatives, carboxylic acids, esters, acid halides, acid amides, halides, aldehydes, alcohols and any other compounds having leaving groups. As these compounds, use can be made of aliphatic, alicyclic, aromatic and heteroaromatic ones. Particularly preferable examples of the polysaccharide derivative to be used in the present invention include ester and carbamate derivatives of polysaccharides having at least 0.1 ester or urethane bond per glucose unit, more preferably ester and carbamate derivatives having an asymmetic center.
The enantiomerically active polymer compound is supported on the carrier preferably in an amount of from 1 to 100% by weight, more preferably from 5 to 60% by weight, and particularly preferably from 15 to 40% by weight, based on the carrier.
The carrier referred to herein includes organic porous substrates and inorganic porous ones, preferably inorganic porous ones. Appropriate examples of the organic porous substrates include polymers comprising polystyrene, polyacrylamide, polyacrylate, etc. Appropriate examples of the inorganic porous substrates include silica, alumina, magnesia, glass, kaolin, titanium oxide, silicates and hydroxyapatite. Silica gel may be cited as a particularly preferable carrier. The particle diameter of the silica gel is from 0.1 xcexcm to 10 mm, preferably from 1 xcexcm to 300 xcexcm, and more preferably from 5 xcexcm to 50 xcexcm and the average pore size thereof is from 10 angstroms to 100 xcexcm, preferably from 50 angstroms to 50,000 angstroms. When silica gel is employed as the carrier, it is preferable to preliminarily surface-coat the silica gel so as to exterminate the effects of the silanol remaining therein, though a non-surface-treated one may be used without any problem.
In the separating agent having supported thereon the enantiomerically active polymer compound of the present invention, the polymer compound may be applied and supported on the carrier through physical adsorption, or may be more firmly immobilized thereto by further forming chemical bonds. These chemical bonds may be formed by, for example, chemical bonds between the carrier and the coated polymer compound, chemical bonds between the polymer compound molecules on the carrier, chemical bonds formed by using a third component, or chemical bonds formed by reactions caused by irradiation of light, radiation such as xcex3-ray, or electromagnetic wave such as micro wave onto the polymer compound on the carrier, or by radical reactions. Furthermore, enantiomerically active polymer compounds as asymmetry recognition agents and enantiomerically inactive polymer compounds may be simultaneously supported on the carrier.
The separating agent for enantiomeric isomers of the present invention is characterized in that when differential scanning calorimetry (DSC) is performed, the polymer compound has an exothermic peak in a differential calorimetric curve obtained in a first heat elevation process before its decomposition temperature is reached; separating agents having such exothermic peaks can have higher asymmetry recognition ability.
It is preferred that DSC measurements are performed in a nitrogen atmosphere. The rate of heat elevation is not particularly limited. It is preferred to perform the DSC measurements at a rate of 0.5 to 100xc2x0 C./min, more preferably 5 to 50xc2x0 C./min. The temperatures at which the DSC measurements are started are not particularly limited. However, it is preferred that the measurements are started at lower temperature than room temperature.
In the DSC measurements, the separating agent containing the polymer compound of the invention, having the exothermic peak(s), may be considered to have an unstable structure in part or on a whole. The method of producing the separating agent containing the polymer compound of the invention is not in particular specified. Conditions of preparation that will influence formation of the unstable structure include, in general for example, heating of the polymer compound, rapid cooling, addition of a plasticizer or another additive and modification caused by introducing a bulky substituent into the polymer compound.
The separating agent of the invention can be obtained by dissolving an enantiomerically active polymer compound in a solvent to obtain a polymer dope, supporting it on a carrier and distilling the solvent out. After the distillation, the product may be heated and then cooled. The product is determined with a differential scanning calorimetry (DSC) to select a polymer compound having an exothermic peak before the decomposition temperature of the polymer compound supported has been reached in the differential calorimetric curve obtained in temperature elevation procedures with the differential scanning calorimetry (DSC) on the separating agent. The supporting step may be carried out by mixing or coating. The distillation may be carried out by heating at a reduced pressure. The supporting step and the distillating step may be repeated.
The separating agent of the invention may be obtained with changed distillating period in time, which may depend on the selected solvent or the distillating temperature.
The solvent to use for supporting the enantiomerically active polymer compounds on the carrier includes any solvent for the used polysaccharide derivative, for example, ketone solvents, ester solvents, ether solvents, amide solvents, imide solvents, hydrocarbon solvents, acid solvents, amine solvents, halogenated solvents, alcohol solvents and nitrile solvents. A single or plural mixed solvents may be used.
The temperature for supporting the enantiomerically active polymer compound on the carrier may be 20xc2x0 C. to 80xc2x0 C.
The distilling period in time after having supported the enantiomerically active polymer compound on the carrier may depend on the solvent used for the supporting step.
The heating treatment may be carried out at any temperature that is not more than the decomposition temperature of the supported enantiomerically active polymer compound, for example preferably at 100xc2x0 C. or lower. The cooling step may be effected rapidly or slowly. The slow cooling may be carried out by allowing the product to stand at a room temperature after the heating. The rapid cooling may be carried out with ice bathing or in a liquid at 0xc2x0 C. or lower such as dry ice-ethanol, dry ice-methanol and liquid nitrogen.
The separating agent of the invention may be used in enantiomeric resolution for example, in chromatography such as gas chromatography, liquid chromatography, thin layer chromatography, super critical chromatography and capillary electrophoresis and then membrane separation. In particular it may be preferably used as a chiral immobilized (stationary) phase of the liquid chromatography. it may be also used for enantiomeric resolution by continuous-wise liquid chromatography such as the simulated moving bed.
A third additive may be used at the supporting step of the enantiomerically active polymer compound on the carrier, for example, any compound not bleeding out during separating use, preferably a polymer such as polystyrene, polycaprolactam, AS resin, poly-methyl methacrylate, polyacetal and polycarbonate.